Development of packaging cell lines for rescuing BAV2 viral vectors

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Abstract

The construction of adenovirus vectors for cloning and foreign gene expression requires
packaging cell lines that can complement missing viral functions caused by sequence deletions
and/or replacement with foreign DNA sequences. In this study, packaging cell lines were
designed to provide in trans the missing bovine adenovirus functions, so that recombinant
viruses could be generated.
Fetal bovine kidney and lUng cells, acquired at the trimester term from a pregnant cow,
were tranfected with both digested wild type BAV2 genomic DNA and pCMV-EI. The plasmid
pCMV-EI was specifically constructed to express El of BAV2 under the control of the
cytomegalovirus enhancer/promoter (CMV). Selection for "true" transformants by continuous
passaging showed no success in isolating immortalised cells, since the cells underwent crisis
resulting in complete cell death. Moreover, selection for G418 resistance, using the same cells,
also did not result in the isolation of an immortalised cell line and the same culture-collapse
event was observed.
The lack of success in establishing an immortalised cell line from fetal tissue prompted us
to transfect a pre-established cell line. We began by transfecting MDBK (Mardin-Dardy bovine
kidney) cells with pCMV-El-neo, which contain the bacterial selectable marker neo gene. A
series of MDBK-derived cell lines, that constitutively express bovine adenoviral (BAV) early
region 1 (El), were then isolated. Cells selected for resistance to the drug G418 were isolated
collectively for full characterisation to assess their suitability as packaging cell lines. Individual
colonies were isolated by limiting dilution and further tested for El expression and efficiency of
DNA uptake. Two cell lines, L-23 and L-24, out of 48 generated foci tested positive for £1
expression using Northern Blot analysis. DNA uptake studies, using both lipofectamine and
calcium phosphate methods, were performed to compare these cells, their parental MDBK cells,
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and the unrelated human 293 cells as a benchmark. The results revealed that the new MDBKderived
clones were no more efficient than MDBK cells in the transient expression of transfected
DNA and that they were inferior to 293 cells, when using lacZ as the reporter gene.
In view of the inherently poor transfection efficiency of MDBK cells and their
derivatives, a number of other bovine cells were investigated for their potential as packaging
cells. The cell line CCL40 was chosen for its high efficiency in DNA uptake and subsequently
transfected with the plasmid vector pCMV El-neo. By selection with the drug G418, two cell
lines were isolated, ProCell 1 and ProCell 2. These cell lines were tested for El expression,
permissivity to BAV2 and DNA uptake efficiency, revealing a DNA uptake efficiency of 37 % ,
comparable to that of CCL40.
Attempts to rescue BAV2 mutants carrying the lacZ gene in place of £1 or £3 were
carried out by co-transfecting wild type viral DNA with either the plasmid pdlElE-Z (which
contains BAV2 sequences from 0% to 40.4% with the lacZ gene in place of the £1 region from
1.1% to 8.25%) or with the plasmid pdlE3-5-Z (which contains BAV2 sequences from 64.8% to
100% with the lacZ gene in place of the E3 region from 75.8% to 81.4%). These cotransfections
did not result in the generation of a viral mutant. The lack of mutant generation
was thought to be caused by the relative inefficiency ofDNA uptake.
Consequently, cosBAV2, a cosmid vector carrying the BAV2 genome, was modified to
carry the neo reporter gene in place of the £3 region from 75.8% to 81.4%. The use of a single
cosmid vector earring the whole genome would eliminate the need for homologous
recombination in order to generate a viral vector. Unfortunately, the transfection of cosBAV2-
neo also did not result in the generation of a viral mutant. This may have been caused by the size
of the £3 deletion, where excess sequences that are essential to the virus' survival might have
been deleted. As an extension to this study, the spontaneous E3 deletion, accidently discovered
in our viral stock, could be used as site of foreign gene insertion.